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    • 专利摘要:
    Moxifloxacin for use in the treatment of spinal muscular atrophy. Moxifloxacin, or a pharmaceutically acceptable salt or solvate thereof, as well as a pharmaceutical composition comprising it, is disclosed for use in the prevention and/or treatment of spinal muscular atrophy (SMA). (Machine-translation by Google Translate, not legally binding)
    公开号:ES2794323A1
    申请号:ES201930436
    申请日:2019-05-17
    公开日:2020-11-17
    发明作者:Allepuz Rubén Artero；Piotr Tadeusz Konieczny；Castel Arturo López；Alonso Manuel Pérez
    申请人:Universitat de Valencia；Fundacion Incliva；
    IPC主号:
    专利说明:

    [0004] TECHNICAL FIELD OF THE INVENTION
    [0006] The present invention relates to moxifloxacin, or a pharmaceutically acceptable salt or solvate thereof, as well as pharmaceutical compositions comprising it, for use in the prevention and / or treatment of spinal muscular atrophy.
    [0008] BACKGROUND OF THE INVENTION
    [0010] Spinal muscular atrophy (SMA, OMIM # 253300) is a rare genetic neuromuscular disease (occurring in 1/6000 births) caused by the loss of amotoneurons from the spinal cord and brainstem nucleus. Five clinical types of spinal muscular atrophy (0, 1, 2, 3, and 4) have been described, ranging from a complete absence of motor function and infant mortality, to minor defects that do not significantly reduce life expectancy. Spinal muscular atrophy is caused by mutations in the SMN1 gene that encodes a 38 kDa protein that is highly expressed in the cytoplasm and in the nucleus.
    [0012] The SMN locus contains an additional, human-specific copy called SMN2. More than 98% of spinal muscular atrophy patients have a homozygous deletion, mutation, or other type of change in the SMN1 gene , and all spinal muscular atrophy patients nevertheless carry one or more copies of the functional SMN2 gene. The SMN1 and SMN2 genes are almost identical, with a difference in the coding region of only one nucleotide: a change from C- (cytidine) to T- (thymidine) in position 6 of exon 7 (Ex7) that causes the exclusion of the exon 7 in most of the SMN2 transcripts ( A7 SMN2), giving rise to a truncated SMN in the C-terminal part and the generation of protein with less stability and activity. However, a small fraction of the SMN2 mRNA includes exon 7 ( FL-SMN2) which generates a fully functional protein. The nucleotide change C to U is in an exonic enhancer region splicing (which term refers to splicing) (exonic splicing enhancer region, by its acronym ESE), in transcripts SMN1 favors a normal splicing of the Ex7. As a consequence, the serine-rich splicing factor SRSF1 (also known as SF2 / ASF) does not effectively recognize its binding site and is not able to remove intron 6. Furthermore, the sequence of ESE (CAGACAA) is transformed into an exonic splicing silencer (ESS) (UAGACAA) and becomes a site recognition of heterologous ribonucleoproteins A1 / A2 (hnRNP A1 / A2). These proteins are known to inhibit splicing, since they operate on ESSs and bind to UAG sites. Reduction factors inhibiting the splicing restores splicing pattern of SMN1, suggesting that there is a competition between enhancers and repressors of the splicing control in places. SMN proteins are dispersed in the cytoplasm, but in the nucleus they aggregate in point-like structures that are close to Cajal bodies, and are called gems, which are aggregates of the SMN protein. Nuclear cells have a direct role in the maturation of small ribonucleoproteins (snRNP) and splicing of pre-mRNA. SMN has been suggested to play a role in the formation of snRNPs in various cell types. Therefore, the appearance of said SMN protein aggregates is directly related to the existence of adequate levels of said protein.
    [0014] Spinal muscular atrophy (SMA) has been described as a curable disease with different therapeutic strategies based on increasing SMN levels, by activating SMN2 transcription and / or seeking to enhance the inclusion of exon 7 in the mature transcripts, resulting in both cases in an increase in the amount of SMN protein. Particularly relevant is the recent commercialization of the drug Spinraza (Nusinersen) by the company Biogen, a drug that consists of a chemically modified antisense oligonucleotide that favors the inclusion of exon 7 of SMN2. However, Spinraza is administered by lumbar puncture, which is invasive and not without risk, and the costs of the drug range from 400,000-600,000 euros per patient per year. The degree of activity of Nusinersen outside the central nervous system and its consequent side effects is also under debate.
    [0016] Although spinal muscular atrophy is considered a neuromotor disease, there is evidence that it affects not only muscles but also the brain, heart, pancreas and even blood vessels. There is also data showing that muscle damage appears before the loss of motor neurons, although this is a subject of active discussion in the scientific community. Therefore, the search for new effective drugs with the capacity to be distributed systemically is an urgent need in the search for treatments against spinal muscular atrophy.
    [0017] BRIEF DESCRIPTION OF THE INVENTION
    [0019] The present invention relates to moxifloxacin, or a pharmaceutically acceptable salt or solvate thereof, as well as to a pharmaceutical composition comprising it, for use in the treatment of spinal muscular atrophy.
    [0021] BRIEF DESCRIPTION OF THE FIGURES
    [0023] Figure 1. Toxicity test. Fibroblasts of the GM03813 line (Corriel Cell Repository) were seeded in 96-well plates with 1.0 x 104 cells per well and incubated for 24 hours. Moxifloxacin was added at various concentrations (1000 µM, 100 µM, 10 µM and 1 µM) in complete Dulbecco's Modified Eagle Medium (DMEM) and the cells were incubated for 48 h. Cell viability was measured with the tetrazolium salt MTS (3- [4,5, dimethylthiazol-2-yl] -5- [3-carboxymethoxy-phenyl] -2- [4-sulfophenyl] -2H-tetrazolium) which was added to each well and incubated for 4 h at 37oC in a chamber moistened with 5% CO 2 . The conversion of MTS in soluble form, by metabolically active cells, was measured by absorbance at 490 nm. The% inhibition of cell viability is represented at each of the moxifloxacin concentrations tested.
    [0025] Figure 2. Quantification of SMN protein levels in fibroblasts derived from spinal muscular atrophy patients, (2 A ) treated with moxifloxacin at the maximum non-toxic concentration: 500 µM, for 72 h. SMN levels were measured by Western blot and the results obtained were compared to those of a sample treated with dimethylsolfoxide solvent (0.5% DMSO, dotted line). (2 B ) Representative images processed with ImageJ of the results obtained by Western blot. Statistical values were calculated using the Student's t test (* p <0.05, ** p <0.01, **** p <0.001).
    [0027] Figure 3. Levels of the SMN2 FL and A7 isoforms in fibroblasts from patients with spinal muscular atrophy treated with moxifloxacin. The fibroblasts were treated with four different concentrations for 48 h. (3 A ) Semi-quantitative analysis of the percentage of splice-in (PSI) in cells treated with 0.5% DMSO compared to cells treated with moxifloxacin 500, 375, 250 and 100 | j M. (3 B ) Representative images processed with ImageJ of the results obtained by splicing on the agarose gel at 2% t. Statistical values were calculated using the Student's t test (* p <0.05, ** p <0.01, **** p <0.0001).
    [0029] Figure 4. Confocal microscopy image of aggregates SMN and Corps Cajal.Se HeLa cells treated with moxifloxacin 500 j M for 72 h the double stained cells for SMN and Coilin. The cores were stained with Hoechst 33258 diluted in mounting medium (1: 10,000). Images were taken with a LSM 800 confocal microscope (Zeiss).
    [0031] Figure 5. Co-localization of SMN and Cajal's Bodies (CB) in HeLa cells after being treated with moxifloxacin. (5 A ) Quantification of SMN aggregates co-localized with Cajal Bodies (CB) from at least 100 HeLa cells treated with moxifloxacin (500 µM) for 72 h. The data obtained were normalized with that of a sample treated with 0.5% solvent. (5 B ) Quantification of the co-localization of the SMN protein with Coilin after treatment with moxifloxacin at different concentrations (ordinate). The results obtained were normalized with those of a sample treated with solvent (DMSO). Statistical values were calculated using the Student's t test (* p <0.05, *** p <0.001).
    [0033] DESCRIPTION OF THE INVENTION
    [0035] The present invention relates to moxifloxacin (1-cyclopropyl-6-fluoro-7 - ((4aS, 7aS) -hexahydro-1H-pyrrolo [3,4-b] pyridin-6 (2H) -yl) -8- methoxy-4-oxo-1,4-dihydroquinoline-3-carboxylic) for use in the prevention and / or treatment of spinal muscular atrophy.
    [0037] Moxifloxacin is an antibiotic with a quinolone structural core that exhibits antibacterial activity.
    [0042] Moxifloxacin has been found to significantly increase SMN protein levels, promoting the inclusion of exon 7 of SMN2 in a concentration-dependent manner, an activity that, as described above, is a treatment and cure route for atrophy spinal muscle (SMA).
    [0044] Moxifloxacin was evaluated in fibroblasts from patients with spinal muscular atrophy, as described in Example 1, by measuring the levels of SMN transcript in the fibroblasts treated with the candidate compound versus the levels of SMN transcript of fibroblasts. vehicle-treated control. On the other hand, moxifloxacin was also evaluated in HeLa cells to determine the existence of SMN aggregates together or co-localized with Cajal's bodies, data indicative of the presence of functional SMN in the organism. For this, immunofluorescence was used, determining the overlap of the signal corresponding to SMN aggregates and the signal corresponding to Cajal's bodies, in HeLa cells treated with moxifloxacin and in cells treated with vehicle.
    [0046] Example 1 (Figures 2 and 3) shows how moxifloxacin increased the levels of the SMN protein in fibroblasts derived from patients with spinal muscular atrophy, causing the inclusion of exon 7 SMN2 in a concentration-dependent manner, and showed the ability to modulate splicing of SMN2 favoring the inclusion of Ex7.
    [0048] Example 2 shows that administration of moxifloxacin to HeLa cells resulted in an almost double increase compared to the DMSO control (Figure 5). These results demonstrate that the increase in SMN levels after treatment with moxifloxacin is beneficial for cells that do not have the function of the SMN1 gene.
    [0050] As seen in the results of the examples of the present invention, moxifloxacin promotes the inclusion of exon 7 of SMN2 in fibroblasts from patients with type II spinal muscular atrophy. Furthermore, the activity of moxifloxacin results in an increase in the expression of the SMN protein and in a greater expression of SMN2 in the form of subcellular structures, or SMN aggregates, a characteristic way in which said protein accumulates under normal conditions. The recovery of normal protein levels could be confirmed by the localization of nuclear SMN aggregates (or gems) next to Cajal Bodies (Figure 4).
    [0051] One embodiment of the invention thus relates to moxifloxacin, or a pharmaceutically acceptable salt or solvate thereof, for use in the prevention and / or treatment of spinal muscular atrophy.
    [0053] There are several types of spinal muscular atrophy depending on the age of the patient and / or the severity of the symptoms.
    [0055] For the purposes of the present invention, a type 0 spinal muscular atrophy is defined as that which affects fetuses. It is the most severe form of the disease and is characterized by poor fetal movement, joint abnormalities, difficulty swallowing, and respiratory failure. It can be fatal before birth and before the first year of life.
    [0056] For the purposes of the present invention, a type 1 spinal muscular atrophy (SMA) is defined as infantile spinal muscular atrophy or Werdnig-Hoffmann disease. It is the most common type of SMA and occurs during birth or in the first few months of life, with developmental delays and problems raising the head, turning, breathing, and swallowing.
    [0058] For the purposes of the present invention, a type 2 spinal muscular atrophy, or intermediate SMA, is defined, that which appears between 7 and 18 months of age, with an inability to walk in children who suffer from it.
    [0060] Furthermore, for the purposes of the present invention, a type 3 spinal muscular atrophy, or juvenile SMA, is defined as that which shows obvious symptoms before the age of three or even during puberty, and which causes problems in standing and walking, worsening said symptoms with age.
    [0062] A type 4 spinal muscular atrophy appears in adults, after the age of 30, and carries only mild or moderate symptoms.
    [0064] One embodiment of the invention relates to moxifloxacin, or a pharmaceutically acceptable salt or solvate thereof, for use in the prevention and / or treatment of spinal muscular atrophy, wherein said spinal muscular atrophy is of type 0, 1, 2, 3 , 4.
    [0066] In one embodiment of the invention, the spinal muscular atrophy is type 0. In another embodiment of the invention, the spinal muscular atrophy is type 1. In another embodiment of the invention, the spinal muscular atrophy is type 2. In another In one embodiment of the invention, the spinal muscular atrophy is type 3. In another embodiment of the invention, the spinal muscular atrophy is type 4.
    [0068] The term "pharmaceutically acceptable salt" refers to any salt which, when administered to the patient, is capable of directly or indirectly providing the described compound.
    [0069] The term "solvate" according to the present invention refers to any form of the active compound, moxifloxacin, that is non-covalently bound to another molecule (usually a polar solvent), and especially refers to hydrates and alcoholates.
    [0071] In one embodiment, the salt is a metal salt. Preferably a salt of an alkali metal or of an alkaline earth metal, more preferably Na +, K + and Ca2 +.
    [0073] In another embodiment, the pharmaceutically acceptable salt is an ammonium salt. Preferably said ammonium salt comprises an ammonium cation substituted with one or more groups independently selected from H, alkyl, alkenyl, alkynyl, hydroxyalkyl, poly (hydroxy) alkyl, cycloalkyl, alkylaryl, aminoaryl, aminoalkyl, aminoalkenyl, aminoalkynyl, arylalkylaminoalkyl, and alkylaminoaryl; or is a cyclic ammonium cation. In another embodiment, the salt is a guanidine salt. In another embodiment, the pharmaceutically salt is a salt of an amino acid. Preferably said amino acid is L-lysine or L-arginine.
    [0075] In one embodiment, the pharmaceutically acceptable salt is a salt of an inorganic acid, preferably a hydrochloride, hydrobromide, sulfate, or phosphate. In another preferred embodiment the pharmaceutically acceptable salt of moxifloxacin for use according to the present invention is a salt of an organic acid, preferably a citrate, oxalate, salicylate, benzoate, acetate, fumarate or maleate.
    [0077] In a preferred embodiment of the invention, the pharmaceutically acceptable salt of moxifloxacin is a salt formed with the carboxylic acid group or with a basic amine of moxifloxacin, or with both groups.
    [0079] In one embodiment the moxifloxacin for use in accordance with the present invention is a basic addition salt. Said salt can also be hydrated.
    [0081] Therefore, a preferred embodiment of the present invention relates to a moxifloxacin hydrochloride salt for use in the prevention and / or treatment of spinal muscular atrophy.
    [0083] Another preferred embodiment of the present invention relates to a moxifloxacin hydrochloride monohydrate salt, for use in the prevention and / or treatment of spinal muscular atrophy.
    [0085] Another embodiment of the invention relates to the use of moxifloxacin, or a pharmaceutically acceptable salt or solvate thereof, for the manufacture of a medicament for use in the treatment of spinal muscular atrophy. Preferably the salt is a hydrochloride.
    [0087] An embodiment of the present invention also relates to a pharmaceutical composition comprising an effective amount of moxifloxacin, or a pharmaceutically acceptable salt or solvate thereof, and at least one excipient or vehicle, for use in the prevention and / or treatment of spinal muscular atrophy.
    [0089] An effective amount, for the purposes of the present invention, is understood as that which provides a therapeutic effect without providing unacceptable toxic effects in the patient. The effective amount or dose of the drug depends on the compound and the condition or disease being treated and on, for example, the age, weight and clinical condition of the patient treated, the mode of administration, the clinical history of the patient, the severity of the disease and the potency of the compound administered.
    [0091] Thus, a preferred embodiment of the present invention relates to a pharmaceutical composition comprising an effective amount of moxifloxacin hydrochloride, for use in the prevention and / or treatment of spinal muscular atrophy.
    [0093] Another preferred embodiment of the present invention relates to a pharmaceutical composition comprising an effective amount of moxifloxacin hydrochloride monohydrate, for use in the prevention and / or treatment of spinal muscular atrophy.
    [0095] Preferably moxifloxacin can be used alone or in conjunction with another active compound. For the purposes of the present invention, an active compound is understood to be a chemical entity or an active principle that exerts therapeutic effects when administered to a human or an animal.
    [0097] Additionally, the present invention describes a method of prevention and / or treatment of spinal muscular atrophy that comprises the administration of an effective amount of moxifloxacin, or a pharmaceutically acceptable salt or solvate thereof, or of a pharmaceutical composition comprising it, to a patient in need. Preferably the pharmaceutically acceptable salt is a hydrochloride.
    [0099] The compositions of the invention include, together with the compounds described in the present invention at least one pharmaceutically acceptable excipient, which can be a carrier or diluent. A diluent is such that it does not modify the biological action of the compound of interest.
    [0101] Said compositions can be included in capsules, tablets, sachets or sachets or any other type of presentation.
    [0103] To make such compositions, conventional techniques for the preparation of pharmaceutical compositions can be used. For example, the compound of interest can be mixed with a vehicle or diluted in a vehicle or contained in a vehicle in the form of an ampoule, capsule, tablet, sachet, sachet or other container. When the carrier serves as a solvent, it can be solid, semi-solid or liquid and act as an excipient or medium for said active compound. The compound of interest can be adsorbed onto a solid granular medium. Some examples of suitable vehicles are water, saline solutions, alcohols, polyethylene glycols, polyhydroxyethoxylated castor oil, peanut oil, olive oil, lactose, terra alba, sucrose, cyclodextrins, amylose, magnesium stearate, talc, gelatin, agar, pectin, acacia, stearic acid, alkyl cellulose ethers, silicon acid, fatty acids, fatty acid amines, monoglycerides and diglycerides of fatty acids, fatty esters of pentaerythrol, polyethylene, hydroxymethylcellulose, and polyvinylpyrrolidone. Likewise, the vehicle or support can include sustained release materials known in the state of the art, such as glyceryl monostearate or diesterate alone or mixed with a wax. The formulations can also include wetting, emulsifying, suspending, preserving, sweetening or flavoring agents. The compositions can be formulated to provide rapid, sustained or delayed release of the active agent after it is administered to the patient using methods known in the art.
    [0105] The pharmaceutical compositions can be sterilized and mixed, if desired, with additional agents, emulsifiers, salt to influence osmotic pressure, buffers and / or coloring substances that do not react adversely with the active compounds.
    [0106] An embodiment of the invention relates to the mode of administration, which can be any mode that effectively transports the compound of interest to the desired site of action, such as oral, rectal, or parenteral, eg, subcutaneous, intravenous, intraurethral, intramuscular. , intranasal or as an ophthalmic solution.
    [0108] Thus, one embodiment of the invention relates to a composition of moxifloxacin, or a pharmaceutically acceptable salt or solvate thereof, for use in accordance with the present invention, wherein said composition is administered orally, rectally, ophthalmic or parenterally. .
    [0110] Preferably, the pharmaceutical composition for use according to the present invention is a tablet, an injectable solution, or an ophthalmic solution.
    [0112] For oral administration, both solid and liquid dosage forms can be prepared. To prepare solid compositions as tablets, the compound of interest is mixed in a formulation with other conventional ingredients such as talc, magnesium stearate, dicalcium phosphate, magnesium aluminum silicate, starch, lactose, acacia, methyl cellulose and functionally similar materials such as pharmaceutical carriers and diluents.
    [0114] Capsules can be prepared by mixing the compound of interest with a pharmaceutically inert solvent and filling the mixture into an appropriately sized hard gelatin. Soft capsules are prepared with machines for encapsulating suspensions of the compound of interest with an acceptable vegetable oil, a light paraffin or an inert oil.
    [0115] Liquid dosage forms such as syrups, elixirs and suspensions can also be prepared. The water-soluble forms can be dissolved in an aqueous vehicle along with sugar, flavorings, and preservatives to form a syrup. An elixir is prepared using a hydroalcoholic vehicle (eg ethanol) with suitable sweeteners such as sugar or saccharin, along with flavoring aromatic agents. Suspensions can be prepared with an aqueous vehicle and the aid of a suspending agent such as acacia, tragacanth, methylcellulose, and the like.
    [0117] For nasal administration, the preparation may contain the compound of interest dissolved or suspended in a liquid vehicle, in particular an aqueous vehicle, for application as an aerosol. The carrier may contain additives such as solubilizing agents, for example propylene glycol, surfactants, absorption enhancers such as lecithin (phosphatidylcholine) or cyclodextrin, or preservatives such as parabens.
    [0119] For ophthalmic applications, the compound of interest is formulated in solutions, suspensions and ointments suitable for use in the eye. The concentrations are usually the same as in preparations for local use.
    [0121] For parenteral application, the use of injectable solutions or suspensions, for intradermal, intramuscular, intravascular and subcutaneous use, are obvious to the person skilled in the art.
    [0122] In addition to the compound of interest, the compositions may include other pharmaceutically acceptable non-toxic diluents and excipients, including vehicles commonly used in pharmaceutical compositions commonly used in humans or animals. The diluent is selected so that it does not affect the biological activity of the composition.
    [0124] Examples of diluents used especially in injectable formulations are organic and inorganic saline solutions, Ringer's solution, dextrose solution and Hank's solution. In addition, the compositions can include additives such as other excipients, adjuvants, non-therapeutic and non-immunogenic stabilizers, and the like.
    [0126] Examples of excipients that can be included in the formulation include, but are not limited to cosolvents, surfactants, oils, humectants, emollients, preservatives, stabilizers, and antioxidants. Any physiologically acceptable buffer can be used, such as Tris or phosphate buffers. Effective amounts of diluents or additives or excipients are those that are effective to obtain a pharmaceutically acceptable formulation in terms of solubility and biological activity.
    [0127] Another embodiment relates to the dosage regimen. The term "unit dose" refers to physically discrete units suitable as unit doses for an individual, eg, mammal, human, dog, cat, rodent, etc. wherein each unit contains a predetermined amount of active material calculated to produce the appropriate therapeutic effect in association with the appropriate diluent, carrier or vehicle.
    [0129] EXAMPLES
    [0131] The examples described below are illustrative in nature and are not intended to limit the scope of the present invention. The methods and materials used in the examples described below are also described.
    [0133] RNA extraction and reverse transcription.
    [0135] The RNA extraction from cells was carried out with the ReliaPrep ™ RNA Cell Miniprep System (Promega), following the manufacturer's instructions. This extraction system includes DNase I treatment and therefore RNA reverse transcription from cells does not include this step.
    [0137] RNA concentration and salt contamination was measured with the NanoDrop spectrophotometer (ThermoFisher). The samples were stored at -80oC. 1 ^ g of total RNA was reverse transcribed using SuperScript III reverse transcriptase (Invitrogen) following the manufacturer's instructions. Genomic DNA contamination was monitored in the RNA samples without the addition of reverse transcriptase.
    [0139] PCR Reactions (Polymerase Chain Reaction)
    [0141] For the quantification of the SMN2 FL and A7 transcripts, Probe qPCR Mix (SolisBiodyne) was used. The reaction was carried out in a multiplex fashion, in which the signal from the SMN2 probe and GAPDH were read in a single reaction. 10 ng of cDNA was used as a standard and the concentration of both probes was 250 nM.
    [0143] The primer sets for the detection of SMN2 FL or A7 isoforms (SEQ ID NO.
    [0144] 1 to 4) were used at a final concentration of 500 nM while the concentration of the primer set used for GAPDH was 150 nM. All reactions were carried out using a Step-One Real-Time PCR thermal cycler (Applied Biosystems). In all cases the experiment included three biological samples and three replicates for each sample.
    [0145] Expression levels were normalized to the reference gene using a 2-AACt method. Primers were obtained from Naryshkin, N. A. et al. Science (2014) 345, 688-693:
    [0147]
    [0149] To obtain the splice-in percentages (PSI), that is, the inclusion percentages of exon 7 of SMN2, semi-quantitative PCR was used with 2x PCR Super Master Mix (Bimake). The final concentration of the primers (SEQ ID NO. 5 and 6) was 250 nM and the image analysis was performed using Image J.
    [0151]
    [0153] Cell culture methods.
    [0155] Fibroblasts derived from SMA patients (GM03813; Corriel Cell Repositories) were cultured at 37oC in cell culture bottles with Dulbecco's Modified Eagle Medium (DMEM; Gibco) standard medium and 4.5 g / ml glucose. The medium was supplemented with penicillin (100 U / ml) and streptomycin (100 mg / ml) [1% P / S; Gibco] and 10% inactivated fetal bovine serum (FBS; Gibco).
    [0157] HeLa cells were cultured under the same conditions except DMEM medium with 1 g / ml of glucose was used.
    [0159] For RNA extraction, 400,000 GM03813 fibroblasts were seeded in 60 mm culture plates in 4 ml of supplemented DMEM medium. The following day the medium was changed and the candidate compounds were added at their maximum non-toxic concentration. Cells were incubated for 48 hours and then washed with 1xPBS and harvested in sterile 1.5 ml tubes. The same was done for the evaluation of the effect of the compounds on the SMN protein level with the difference that 1,000,000 fibroblasts were used in a 100 mm cell culture plate and that the cells were harvested after 2 days with a RIPA protein extraction buffer supplemented with a protease inhibitor cocktail (cOmplete; Roche). For immunostaining, 40,000 HeLa cells were seeded in a 400 µl 24-well plate with supplemented DMEM medium followed by change of medium with treatment of the corresponding candidate compound. The cells were incubated for 72 h with the candidate compounds and fixed in 4% paraformaldehyde for 10 minutes. All cell cultures were incubated at 37oC with 5% CO 2 .
    [0161] Example 1: Evaluation of moxifloxacin in SMA fibroblasts.
    [0163] Moxifloxacin was evaluated in fibroblasts derived from patients with type II spinal muscular atrophy. The dose used in the study was determined experimentally in normal fibroblasts.
    [0165] The toxicity test was carried out using the CellTiter 96® Aqueous Non-Radioactive Cell Proliferation Assay (Promega). Fibroblasts of the GM03814 line were seeded in 96-well plates with 1.0 x 104 cells per well and incubated for 24 hours. Moxifloxacin was added at various concentrations in complete DMEM medium and the cells were incubated for 48 h. Cell viability was measured with the MTS tetrazolium salt that was added to each well and incubated for 4 h at 37oC in a chamber moistened with 5% CO 2 . The conversion of MTS in soluble form, by metabolically active cells, was measured by absorbance at 490 nm.
    [0167] The inhibition threshold for cell viability was established at 10% (Figure 1) and the moxifloxacin concentration in subsequent studies did not exceed this value.
    [0169] Fibroblasts derived from SMA type II patients were incubated with moxifloxacin at the maximum non-toxic concentration previously established for 72 h.
    [0171] 50 ^ g of protein extracted from cells treated with moxifloxacin were separated by electrophoresis with acrylamide / bis-acrylamide gels SDS-PAGE with the Mini-protean Electrophoresis System (Bio-Rad). After carrying out the electrophoresis, the proteins were transferred to a nitrocellulose membrane (GE Healthcare) using a semi-dry electro-transfer system (Bio-Rad) for one hour with a constant voltage of 15V. The membranes were incubated with a blocking solution (5% skim milk in PBST, 1x PBS, 0.1% Tween 20) for at least 1h at room temperature.
    [0172] They were then incubated at 4oC overnight with the SMN primary antibody blocking solution, MANSMA1 (11F3; Developmental Studies Hybridoma Bank) and subsequently incubated with an anti-mouse peroxidase-IgG conjugated secondary antibody (Sigma) for 1 hour at room temperature. p-actin was used as a loading control (ACTB in Figure 2B), using an antibody against p-actin and a secondary peroxidase-conjugated rabbit anti-ilgG conjugate.
    [0174] Chemiluminescence was detected using SuperSignal West Femto Maximum Sensitivity Substrate (Pierce) and recorded with ImageQuant LAS 4000 (GE Healthcare).
    [0176] The levels of SMN measured by Western blot and the results obtained were compared to those of a sample treated with solvent (DMSO 0.5%) corresponding to the dotted line of figure 2A. In addition, representative images of the results obtained in the blot as shown in figure 2B. Statistical values were calculated using the Student's t-test (* p <0.05, ** p <0.01, **** p <0.001). In said Figures 2A and 2B it can be seen that moxifloxacin produced a significant increase in SMN protein levels compared to the control with DMSO.
    [0178] Their effect on the SMN2 transcript was then evaluated. The selected SMA fibroblast line has 3 copies of the SMN2 gene and a homozygous deletion of Ex7-Ex8 in SMN1, and thus only transcripts of the SMN2 gene were measured .
    [0180] Moxifloxacin increases the levels of SMN protein and changes the ratio of the isoforms SMN2FL and A7, promoting the inclusion of exon 7 in the final transcript, as can be observed in Figure 3B. In this sense, the FL isomorph, which includes exon 7, increases as a function of concentration, while the A7 isomorph, an isomorph that excludes said exon, decreases.
    [0182] The semi-quantitative analysis of the percentage of splice-in (PSI), that is, of inclusion, of exon 7 of SMN2 showed an increase of 63% in cells treated with solvent to 90% in cells treated with moxifloxacin 500 µM (Figure 3A).
    [0184] Example 2: Evaluation of moxifloxacin in HeLa cells
    [0186] Moxifloxacin showed the ability to modulate the result of SMN2 splicing , favoring the inclusion of Ex7. It was then checked whether the increase in the SMN2 FL transcript was translated into stable and functional SMN protein.
    [0187] For this, HeLa cells were treated with 500 µM moxifloxacin for 72 h and the cells were double stained for SMN and Coilin. Coilin is a molecular component of the Cajal Bodies (CB) and is responsible for the formation of a bridge between SMN and CB that is only formed with complete and functional SMN protein.
    [0189] For this, HeLa cells were seeded in 24-well plates, 400 µL of complete DMEM, with 40,000 cells per well and incubated overnight at 37oC. The following day the cells were treated with DMSO (0.5%) and moxifloxacin at four different concentrations (500, 375, 250 and 50 µM; diluted with 0.5% DMSO). The cells were incubated for 72h and fixed with 4% paraformaldehyde for 10 minutes. The membrane was permeabilized with 1x PBST (1x PBS, 0.3% Triton X-100) for 15 min with subsequent incubation in blocking buffer (1x PBST, 4% normal goat serum) for 1 h at room temperature. Primary monoclonal anti-SMN2 (mouse, Millipore) and polyclonal anti-Coilin (rabbit, Invitrogen) antibodies were diluted with blocking buffer (1: 400) and incubated overnight at 4 ° C. Secondary anti-mouse antibodies conjugated with Rhodamine and anti-rabbit conjugated with fluorescein isothiocyanate (FITC), were diluted with blocking buffer (1: 400) and incubated at 4 ° C overnight. The cores were stained with Hoechst 33258 diluted in mounting medium (1: 10,000). Images were taken with a LSM 800 confocal microscope (Zeiss). The co-location analysis of SMN and Cajal Bodies aggregates and the Manders Overlap Coefficient calculations were carried out with the ZEN software .
    [0191] It was observed that HeLa cells treated with moxifloxacin significantly increased the number of SMN aggregates, as well as the CBs (Cajal Bodies), as can be seen in the images in Figure 4 The number of SMN aggregates co-localized with CBs was calculated and found to double compared to the control treated with solvent only (Figure 5A). To confirm this observation, a co-localization analysis was carried out using the ZEN software that allows the visualization and quantification of the overlap between the detected SMN and Coilin fluorescence.
    [0193] A significant increase in co-localization between the two proteins was observed after treatment. The analysis carried out for different concentrations of moxifloxacin showed that at 250 | j M significantly increases the functional levels of SMN in HeLa cells (Figure 5B). Moxifloxacin was shown to improve the inclusion of Ex7 SMN2 and increase the levels of functional SMN protein, showing the utility of said compound in the treatment of SMA.
    权利要求:
    Claims (10)
    [1]
    1. Moxifloxacin, or a pharmaceutically acceptable salt or solvate thereof, for use in the prevention and / or treatment of spinal muscular atrophy.
    [2]
    2. Moxifloxacin, or a pharmaceutically acceptable salt or solvate thereof, for use according to claim 1, wherein the spinal muscular atrophy is of type 0, 1, 2, 3 or 4.
    [3]
    3. Moxifloxacin, or a pharmaceutically acceptable salt or solvate thereof, for use according to any of claims 1 or 2, wherein the pharmaceutically acceptable salt of moxifloxacin is moxifloxacin hydrochloride.
    [4]
    4. A pharmaceutical composition comprising an effective amount of moxifloxacin, or a pharmaceutically acceptable salt or solvate thereof, and at least one excipient or vehicle, for use in the prevention and / or treatment of spinal muscular atrophy.
    [5]
    5. A pharmaceutical composition for use according to claim 4, wherein the spinal muscular atrophy is of type 0, 1, 2, 3 or 4.
    [6]
    6. A pharmaceutical composition for use according to any of claims 4 or 5, wherein the pharmaceutically acceptable salt of moxifloxacin is moxifloxacin hydrochloride.
    [7]
    7. A pharmaceutical composition for use according to any of claims 4 to 6, wherein said composition is administered orally, ophthalmic or parenterally.
    [8]
    8. A pharmaceutical composition for use according to any one of claims 4 to 6, wherein said composition is a tablet.
    [9]
    9. A pharmaceutical composition for use according to any one of claims 4 to 6, wherein said composition is an injectable solution.
    [10]
    10. A pharmaceutical composition for use according to any of claims 4 to 6, wherein said composition is an ophthalmic solution.
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    WO2020234496A1|2020-11-26|
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    PCT/ES2020/070311| WO2020234496A1|2019-05-17|2020-05-14|Moxifloxacin for use in the treatment of spinal muscular atrophy|
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